Discovery of a novel and highly selective JAK3 inhibitor as a potent hair growth promoter

JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC50 of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-024-05144-4.


S1 -Table2
Binding affinity and H-bonding interactions of ligand molecule (MJ-04) in the hinge region of the kinase domain of JAK1, JAK2, and JAK3 proteins.meta-Chloroperbenzoic acid (m-CPBA) (1.5 mmol) was added portion wise to an ice-cooled solution of 1 (1.0 mmol) in dichloromethane, and the resulting reaction mixture was stirred at 25 o C for 2 h.After that completion of the reaction was monitored with the help of thin-layer chromatography, and then the reaction mixture was washed with saturated solution of sodium bicarbonate, extracted it with dichloromethane, and concentrated in-vaccuo to obtain solid compound 2, which was used for the next step without further purification.To the solution of 2 in tetrahydrofuran, cyclopropyl amine (2.0 equiv) was added, and resulting reaction mixture was stirred at room temperature for 2 h and then solvents were removed in vaccuo after that residue was absorbed onto celite and purified chromatographically on silica gel with ethyl acetate/hexane system to obtained pure compounds 3. Next, we started synthesis of compound 6 that began with a Suzuki cross coupling reaction between 5-bromo-7-azaindole and 4-fluoro phenylboronic acid that gave 5-aryl substituted 7-azaindole (6) in good yield (81 %).Iodination of 6 in presence of iodine, potassium hydroxide gave (7) which on further treated with Boc anhydride gave (8) in good yield (84 %).Next by using tetrakis (triphenylphosphine)-palladium (0) (3 mol %) and intermediates (8) (1.00 mmol) were placed under argon atmosphere in a dry screw-cap vessel with septum.Then, 5 mL of dry dioxane was added, and the mixture was degassed with argon.Dry triethylamine (10.0 mmol, 10.0 equiv) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.50 mmol, 1.50 equiv) were successively added to the mixture, which was stirred at 80 °C (preheated oil bath) for 3-4 h to obtain 9 (monitored by TLC).Then, after cooling at 25 o C (water bath), tetrakis (triphenylphosphine)-palladium (0) (3 mol %), 5 mL of dry methanol, 1.00 mmol of compound 3, and cesium carbonate (2.50 mmol, 2.50 equiv) were successively added, and the mixture was stirred at 100 °C (preheated oil bath) for 35-49 h.Then, after cooling at 25 o C (water bath), the solvents were removed in vaccuo, and the residue was absorbed onto Celite and purified chromatographically on silica gel 230-400 with dichloromethane-methanol aqueous ammonia (isocratic or stepwise gradient).The obtained bis(hetero)aryls can be further purified by suspending them in dichloromethane, sonication in an ultrasound bath for 0.5-1.0h, filtration, and drying in vaccuo overnight for 12 h to obtain the compound MJ04.Group 1-No treatment, Group 2-treated with 0.5% testosterone, Group 3 -treated with 0.5% testosterone followed by vehicle treatment after 1 h, Group 4 -treated with 0.5% testosterone followed by Tofacitinib citrate (0.8 mg/Kg) treatment after 1 h, Group 5 -treated with 0.5% testosterone followed by Tofacitinib citrate (0.08 mg/Kg) treatment after 1 h, Group 6-treated with 0.5% testosterone followed by MJ04 (0.08 mg/Kg) treatment after 1 h, Group 7-treated with 0.5% testosterone followed by MJ04 (0.04 mg/Kg) treatment after 1 h, Group 8-treated with 0.5% testosterone followed by MJ04 (0.016 mg/Kg) treatment after 1 h, Group 9 -treated with 0.5% testosterone followed by Baricitinib (0.1mg/Kg) after 1 h, Group 10 -treated with 0.5% testosterone followed by Baricitinib (0.04 mg/Kg) after 1 h, and Group 11 -treated with 0.5% testosterone followed by Baricitinib (0.02 mg/Kg) after 1 h.Group 1-No treatment, Group 2-treated with 0.5% testosterone, Group 3 -treated with 0.5% testosterone followed by vehicle treatment after 1 h, Group 4 -treated with 0.5% testosterone followed by Tofacitinib citrate (0.8 mg/Kg) treatment after 1 h, Group 5 -treated with 0.5% testosterone followed by Tofacitinib citrate (0.08 mg/Kg) treatment after 1 h, Group 6-treated with 0.5% testosterone followed by MJ04 (0.08 mg/Kg) treatment after 1 h, Group 7-treated with 0.5% testosterone followed by MJ04 (0.04 mg/Kg) treatment after 1 h, Group 8-treated with 0.5% testosterone followed by MJ04 (0.016 mg/Kg) treatment after 1 h, Group 9 -treated with 0.5% testosterone followed by Baricitinib (0.1mg/Kg) after 1 h, Group 10 -treated with 0.5% testosterone followed by Baricitinib (0.04 mg/Kg) after 1 h, and Group 11 -treated with 0.5% testosterone followed by Baricitinib (0.02 mg/Kg) after 1 h.s This score is typically in the range from −5 to 5. The higher the score is, the higher the probability is that the molecule is a NP.

Spectral
Lipinski Rule Accepted q s MW ≤ 500; logP ≤ 5; Hacc ≤ 10; Hdon ≤ 5 s If two properties are out of range, a poor absorption or permeability is possible, one is acceptable.

Fig S2A :
Fig S2A: Schematic representation flow regarding selection of compounds against the target Fig 2SC:-1 H -NMR of MJ04

Fig S5 :
Fig S5: Evaluation of toxicity of MJ04 and tofacitinib in splenocytes by MTT assay.

s
Natural product-likeness score.

1. Physicochemical Property Property Value Comment
s MCE-18≥45 is considered a suitable value.
Estrogen receptor ligand-binding domain s Category 1: actives ; Category 0: inactives; s The output value is the probability of being active.The output value is the probability of being active.Heat shock factor response element s Category 1: actives ; Category 0: inactives; s The output value is the probability of being active.Mitochondrial membrane potential s Category 1: actives ; Category 0: inactives; s The output value is the probability of being active.The output value is the probability of being active.